STRUCTURE OF AN RNA HAIRPIN LOOP WITH A 5'-CGUUUCG-3' LOOP MOTIF BY HETERONUCLEAR NMR-SPECTROSCOPY AND DISTANCE GEOMETRY

Structural features of a 19-nucleotide RNA hairpin loop (5'-GGCGUACGUU UCGUACGCC-3'), a loop motif which occurs in eukaryotic 18S rRNA, have been derived using multidimensional heteronuclear NMR spectroscopy in combination with local conformational analysis and torsion angle dista nce geometry followed by restrained energy minimization. A method to o btain both the (3)J(C4'P3') and (3)J(C4'P5') coupling constants from a set of spin-echo difference constant time HSQC spectra is introduced, and it is shown how these couplings can be assigned to the backbone a ngles beta and epsilon. A total of 280 distance constraints as well as 132 home-and heteronuclear three-bond scalar coupling constants were derived from the NMR data. The structure which has been determined is a pentaloop rather than a triloop with no base pairing between G8 and C12. G8 is pointed to the minor groove where it forms a base triplet w ith C7-G13 that is further stabilized by hydrogen bonding to the 2'-hy droxyl group of C7. C12 is directed to the major groove where its conf ormation is stabilized by hydrogen bonding between O2 and HO2'. The NM R data suggest two possible, interconverting conformations with stacki ng of bases U10-G8 or U11-C7. Overall, the loop provides a variety of interaction sites for RNA or protein interactions.